PEG: The Magic Bullet for Biophysical Analysis of Highly Aggregating Small Molecules in Aqueous Solutions.

Biophysical research plays a crucial role in drug discovery, but many druglike molecules are poorly soluble and prone to aggregation, making their analysis challenging and susceptible to artifacts. To address this issue, we propose an approach that uses poly(ethylene glycol) (PEG) as an excipient in aqueous buffers to reduce the propensity of small molecules to aggregate. We show how PEG allows us to measure the thermodynamics of a complex formed by a heterobifunctional Small Molecule (hSM) that brings two proteins together. Our model accounts for all of the equilibrium states of the small molecule in solution, resulting in more precise parameters for describing how the proteins and the ligand interact. These precise parameters are important for designing better lead molecules.

Microsecond Motion of the Bacterial Transporter EmrE in Lipid Bilayers.

The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F4-TPP+), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure 15N rotating-frame spin-lattice relaxation (R1ρ) rates of F4-TPP+-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and 1H-detected 15N spin-lock experiments under 55 kHz MAS, we measured 15N R1ρ rates site-specifically. Many residues show spin-lock field-dependent 15N R1ρ relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s-1 at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.

High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport.

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN-F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

From Angstroms to Nanometers: Measuring Interatomic Distances by Solid-State NMR.

Internuclear distances represent one of the main structural constraints in molecular structure determination using solid-state NMR spectroscopy, complementing chemical shifts and orientational restraints. Although a large number of magic-angle-spinning (MAS) NMR techniques have been available for distance measurements, traditional 13C and 15N NMR experiments are inherently limited to distances of a few angstroms due to the low gyromagnetic ratios of these nuclei. Recent development of fast MAS triple-resonance 19F and 1H NMR probes has stimulated the design of MAS NMR experiments that measure distances in the 1-2 nm range with high sensitivity. This review describes the principles and applications of these multiplexed multidimensional correlation distance NMR experiments, with an emphasis on 19F- and 1H-based distance experiments. Representative applications of these long-distance NMR methods to biological macromolecules as well as small molecules are reviewed.

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.

Structure and drug binding of the SARS-CoV-2 envelope protein transmembrane domain in lipid bilayers.

An essential protein of the SARS-CoV-2 virus, the envelope protein E, forms a homopentameric cation channel that is important for virus pathogenicity. Here we report a 2.1-Å structure and the drug-binding site of E's transmembrane domain (ETM), determined using solid-state NMR spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow pore. The protein deviates from the ideal α-helical geometry due to three phenylalanine residues, which stack within each helix and between helices. Together with valine and leucine interdigitation, these cause a dehydrated pore compared with the viroporins of influenza viruses and HIV. Hexamethylene amiloride binds the polar amino-terminal lumen, whereas acidic pH affects the carboxy-terminal conformation. Thus, the N- and C-terminal halves of this bipartite channel may interact with other viral and host proteins semi-independently. The structure sets the stage for designing E inhibitors as antiviral drugs.

Structure and Drug Binding of the SARS-CoV-2 Envelope Protein in Phospholipid Bilayers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Successful development of vaccines and antivirals against SARS-CoV-2 requires a comprehensive understanding of the essential proteins of the virus. The envelope (E) protein of SARS-CoV-2 assembles into a cation-selective channel that mediates virus budding, release, and host inflammation response. E blockage reduces virus pathogenicity while E deletion attenuates the virus. Here we report the 2.4 Å structure and drug-binding site of E's transmembrane (TM) domain, determined using solid-state nuclear magnetic resonance (NMR) spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow central pore. The middle of the TM segment is distorted from the ideal a-helical geometry due to three regularly spaced phenylalanine residues, which stack within each helix and between neighboring helices. These aromatic interactions, together with interhelical Val and Leu interdigitation, cause a dehydrated pore compared to the viroporins of influenza and HIV viruses. Hexamethylene amiloride and amantadine bind shallowly to polar residues at the N-terminal lumen, while acidic pH affects the C-terminal conformation. These results indicate that SARS-CoV-2 E forms a structurally robust but bipartite channel whose N- and C-terminal halves can interact with drugs, ions and other viral and host proteins semi-independently. This structure establishes the atomic basis for designing E inhibitors as antiviral drugs against SARS-CoV-2.

Two-dimensional 19F-13C correlation NMR for 19F resonance assignment of fluorinated proteins.

19F solid-state NMR is an excellent approach for measuring long-range distances for structure determination and for studying molecular motion. For multi-fluorinated proteins, assignment of 19F chemical shifts has been traditionally carried out using mutagenesis. Here we show 2D 19F-13C correlation experiments that allow efficient assignment of the 19F chemical shifts. We have compared several rotational-echo double-resonance-based pulse sequences and 19F-13C cross polarization (CP) for 2D 19F-13C correlation. We found that direct transferred-echo double-resonance (TEDOR) transfer from 19F to 13C and vice versa outperforms out-and-back coherence transfer schemes. 19F detection gives twofold higher sensitivity over 13C detection for the 2D correlation experiment. At MAS frequencies of 25-35 kHz, double-quantum 19F-13C CP has higher coherence transfer efficiencies than zero-quantum CP. The most efficient TEDOR transfer experiment has higher sensitivity than the most efficient double-quantum CP experiment. We demonstrate these 2D 19F-13C correlation experiments on the model compounds t-Boc-4F-phenylalanine and GB1. Application of the 2D 19F-13C TEDOR correlation experiment to the tetrameric influenza BM2 transmembrane peptide shows intermolecular 13C-19F cross peaks that indicate that the BM2 tetramers cluster in the lipid bilayer in an antiparallel fashion. This clustering may be relevant for the virus budding function of this protein.

Atomic structures of closed and open influenza B M2 proton channel reveal the conduction mechanism.

The influenza B M2 (BM2) proton channel is activated by acidic pH to mediate virus uncoating. Unlike influenza A M2 (AM2), which conducts protons with strong inward rectification, BM2 conducts protons both inward and outward. Here we report 1.4- and 1.5-Å solid-state NMR structures of the transmembrane domain of the closed and open BM2 channels in a phospholipid environment. Upon activation, the transmembrane helices increase the tilt angle by 6° and the average pore diameter enlarges by 2.1 Å. BM2 thus undergoes a scissor motion for activation, which differs from the alternating-access motion of AM2. These results indicate that asymmetric proton conduction requires a backbone hinge motion, whereas bidirectional conduction is achieved by a symmetric scissor motion. The proton-selective histidine and gating tryptophan in the open BM2 reorient on the microsecond timescale, similar to AM2, indicating that side chain dynamics are the essential driver of proton shuttling.

Elucidating Relayed Proton Transfer through a His-Trp-His Triad of a Transmembrane Proton Channel by Solid-State NMR.

Proton transfer through membrane-bound ion channels is mediated by both water and polar residues of proteins, but the detailed molecular mechanism is challenging to determine. The tetrameric influenza A and B virus M2 proteins form canonical proton channels that use an HxxxW motif for proton selectivity and gating. The BM2 channel also contains a second histidine (His), H27, equidistant from the gating tryptophan, which leads to a symmetric H19xxxW23xxxH27 motif. The proton-dissociation constants (pKa's) of H19 in BM2 were found to be much lower than the pKa's of H37 in AM2. To determine if the lower pKa's result from H27-facilitated proton dissociation of H19, we have now investigated a H27A mutant of BM2 using solid-state NMR. 15N NMR spectra indicate that removal of the second histidine converted the protonation and tautomeric equilibria of H19 to be similar to the H37 behavior in AM2, indicating that the peripheral H27 is indeed the origin of the low pKa's of H19 in wild-type BM2. Measured interhelical distances between W23 sidechains indicate that the pore constriction at W23 increases with the H19 tetrad charge but is independent of the H27A mutation. These results indicate that H27 both accelerates proton dissociation from H19 to increase the inward proton conductance and causes the small reverse conductance of BM2. The proton relay between H19 and H27 is likely mediated by the intervening gating tryptophan through cation-π interactions. This relayed proton transfer may exist in other ion channels and has implications for the design of imidazole-based synthetic proton channels.

High-Sensitivity Detection of Nanometer 1H-19F Distances for Protein Structure Determination by 1H-Detected Fast MAS NMR.

Protein structure determination by solid-state NMR requires the measurement of many interatomic distances through dipole-dipole couplings. To obtain multiple long-range distance restraints rapidly and with high sensitivity, here we demonstrate a new 1H-detected fast magic-angle-spinning NMR technique that yields many long distances in a two-dimensional (2D)-resolved fashion. The distances are measured up to ∼15 Å, with an accuracy of better than 10%, between 1H and 19F, two nuclear spins that have the highest gyromagnetic ratios. Exogenous fluorines are sparsely introduced into the aromatic residues of the protein, which is perdeuterated and back-exchanged to give amide protons. This 1H-19F distance experiment, termed 2D heteronuclear single-quantum coherence rotational-echo double-resonance (HSQC-REDOR), is demonstrated on the singly fluorinated model protein, GB1. We extracted 33 distances between 5-19F-Trp43 and backbone amide protons, using 2D spectral series that were measured in less than 3 days. Combining these 1H-19F distance restraints with 13C-19F distances and chemical shifts, we calculated a GB1 structure with a backbone root-mean-square deviation of 1.73 Å from the high-resolution structure. This 1H-detected 1H-19F distance technique promises to provide a highly efficient tool for constraining the three-dimensional structures of proteins and protein-ligand complexes, with not only precise and fast measurements but also access to truly long-range distances.

Long-term impact of fecal transplantation in healthy volunteers.

BACKGROUND: Fecal microbiota transplantation (FMT) has been recently approved by FDA for the treatment of refractory recurrent clostridial colitis (rCDI). Success of FTM in treatment of rCDI led to a number of studies investigating the effectiveness of its application in the other gastrointestinal diseases. However, in the majority of studies the effects of FMT were evaluated on the patients with initially altered microbiota. The aim of our study was to estimate effects of FMT on the gut microbiota composition in healthy volunteers and to monitor its long-term outcomes. RESULTS: We have performed a combined analysis of three healthy volunteers before and after capsule FMT by evaluating their general condition, adverse clinical effects, changes of basic laboratory parameters, and several immune markers. Intestinal microbiota samples were evaluated by 16S rRNA gene and shotgun sequencing. The data analysis demonstrated profound shift towards the donor microbiota taxonomic composition in all volunteers. Following FMT, all the volunteers exhibited gut colonization with donor gut bacteria and persistence of this effect for almost ∼1 year of observation. Transient changes of immune parameters were consistent with suppression of T-cell cytotoxicity. FMT was well tolerated with mild gastrointestinal adverse events, however, one volunteer developed a systemic inflammatory response syndrome. CONCLUSIONS: The FMT leads to significant long-term changes of the gut microbiota in healthy volunteers with the shift towards donor microbiota composition and represents a relatively safe procedure to the recipients without long-term adverse events.

Rapid measurement of long-range distances in proteins by multidimensional 13C-19F REDOR NMR under fast magic-angle spinning.

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are 13C-15N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~ 5 Å due to the low gyromagnetic ratios of 15N and 13C. Here we present a robust 2D 13C-19F REDOR experiment to measure multiple distances to ~ 10 Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The 13C-19F REDOR sequence is combined with 2D 13C-13C correlation to resolve multiple distances in highly 13C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved 13C spectra, the deleterious effect of the large 19F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D 13C-13C resolved 13C-19F REDOR technique on 13C, 15N-labeled GB1. A 5-19F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-19F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical 19F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D 13C-19F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~ 10 Å range.

Fast Magic-Angle-Spinning 19F Spin Exchange NMR for Determining Nanometer 19F-19F Distances in Proteins and Pharmaceutical Compounds.

Internuclear distances measured using NMR provide crucial constraints of three-dimensional structures but are often restricted to about 5 Å due to the weakness of nuclear-spin dipolar couplings. For studying macromolecular assemblies in biology and materials science, distance constraints beyond 1 nm will be extremely valuable. Here we present an extensive and quantitative analysis of the feasibility of 19F spin exchange NMR for precise and robust measurements of interatomic distances up to 1.6 nm at a magnetic field of 14.1 T, under 20-40 kHz magic-angle spinning (MAS). The measured distances are comparable to those achievable from paramagnetic relaxation enhancement but have higher precision, which is better than ±1 Å for short distances and ±2 Å for long distances. For 19F spins with the same isotropic chemical shift but different anisotropic chemical shifts, intermediate MAS frequencies of 15-25 kHz without 1H irradiation accelerate spin exchange. For spectrally resolved 19F-19F spin exchange, 1H-19F dipolar recoupling significantly speeds up 19F-19F spin exchange. On the basis of data from five fluorinated synthetic, pharmaceutical, and biological compounds, we obtained two general curves for spin exchange between CF groups and between CF3 and CF groups. These curves allow 19F-19F distances to be extracted from the measured spin exchange rates after taking into account 19F chemical shifts. These results demonstrate the robustness of 19F spin exchange NMR for distance measurements in a wide range of biological and chemical systems.

Protonation equilibria and pore-opening structure of the dual-histidine influenza B virus M2 transmembrane proton channel from solid-state NMR.

The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2 contains a second titratable histidine, His-27, in the tetrameric transmembrane domain that forms a reverse WXXXH motif with the gating tryptophan. To understand how His-27 affects the proton conduction property of BM2, we have used solid-state NMR to characterize the pH-dependent structure and dynamics of His-27. In cholesterol-containing lipid membranes mimicking the virus envelope, 15N NMR spectra show that the His-27 tetrad protonates with higher pKa values than His-19, indicating that the solvent-accessible His-27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His-19. AM2 is inhibited by the amantadine class of antiviral drugs, whereas BM2 has no known inhibitors. We measured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe-5. The interhelical 19F-19F distances show a bimodal distribution of a short distance of 7 Å and a long distance of 15-20 Å, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels.

Optimization of a ß-sheet-cap for long loop closure.

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable ß-sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two ß-strands connected by varying loop lengths and composition (an example sequence: RWITVTI - loop - KKIRVWE). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: RWITVTI-(GGGGKK)2 GGGG-KKIRVWE) remaining folded (ΔGU = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: RWITVRIW-(GGGGKK)2 GGGG-WKTIRVWE) supports a remarkably stable ß-sheet (ΔGU = 6.3 kJ/mol at 295 K, Tm = ∼55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.

Nascent Hairpins in Proteins: Identifying Turn Loci and Quantitating Turn Contributions to Hairpin Stability.

Many factors influence the stability of hairpins that could appear as foldons in partially folded states of proteins; of these, the propensity of certain amino acid sequences to favor conformations that serve to align potential ß-strands for antiparallel association is likely the dominant feature. Quantitating turn propensities is viewed as the first step in developing an algorithm for locating nascent hairpins in protein sequences. Such nascent hairpins can serve to accelerate protein folding or, if they represent structural elements that differ from the final folded state, as kinetic traps. We have measured these "turn propensities" for the two most common turn types using a series of model peptide hairpins with four- and six-residue loops connecting the associated ß-strands. Loops of four to six residues with specific turn sequences containing only natural l-amino acids and glycine can provide as much as 15 kJ/mol of hairpin stabilization versus loops lacking the defined turn loci. Single-site mutations within some of the optimal connecting loops can have ΔΔG effects as large as 9-10 kJ/mol on hairpin stability. In contrast to the near universal II'/I' turns of model hairpins, a number of hairpin-supporting XZZG sequence ß-turns with αR and/or γR configurations at the ZZ unit were found. A series of turn replacements (four-residue ß-turns replaced by sequences that favor five- and six-residue reversing loops) using identical strands in our model systems have confirmed that several sequences have intrinsic turn propensities that could favor ß-strand association in a non-native strand register and thus serve as kinetic traps. These studies also indicate that aryl residues immediately flanking a turn sequence can alter relative turn propensities by as much as 9-11 kJ/mol and will need to be a part of any nascent hairpin recognition algorithm.

Aryl-aryl interactions in designed peptide folds: Spectroscopic characteristics and optimal placement for structure stabilization.

We have extended our studies of Trp/Trp to other Aryl/Aryl through-space interactions that stabilize hairpins and other small polypeptide folds. Herein we detail the NMR and CD spectroscopic features of these types of interactions. NMR data remains the best diagnostic for characterizing the common T-shape orientation. Designated as an edge-to-face (EtF or FtE) interaction, large ring current shifts are produced at the edge aryl ring hydrogens and, in most cases, large exciton couplets appear in the far UV circular dichroic (CD) spectrum. The preference for the face aryl in FtE clusters is W ≫ Y ≥ F (there are some exceptions in the Y/F order); this sequence corresponds to the order of fold stability enhancement and always predicts the amplitude of the lower energy feature of the exciton couplet in the CD spectrum. The CD spectra for FtE W/W, W/Y, Y/W, and Y/Y pairs all include an intense feature at 225-232 nm. An additional couplet feature seen for W/Y, W/F, Y/Y, and F/Y clusters, is a negative feature at 197-200 nm. Tyr/Tyr (as well as F/Y and F/F) interactions produce much smaller exciton couplet amplitudes. The Trp-cage fold was employed to search for the CD effects of other Trp/Trp and Trp/Tyr cluster geometries: several were identified. In this account, we provide additional examples of the application of cross-strand aryl/aryl clusters for the design of stable ß-sheet models and a scale of fold stability increments associated with all possible FtE Ar/Ar clusters in several structural contexts. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 337-356, 2016.

An improved capping unit for stabilizing the ends of associated ß-strands.

Understanding protein beta structures has been hindered by the challenge of designing small, well-folded ß-sheet systems. A ß-capping motif was previously designed to help solve this problem, but not without limitations, as the termini of this ß-cap were not fully available for chain extension. Combining Coulombic side chain attractions with a Trp/Trp edge-to-face interaction we produced a new capping motif that provided greater ß-sheet stability. This stability was maintained even in systems lacking a turn locus with a high propensity for chain direction reversal. The Coulombic cap was shown to improve ß-sheet stability in a number of difficult systems, hence providing an additional tool for protein structure and folding studies.